Advances and Challenges in Targeting TGF-β Isoforms for Therapeutic Intervention of Cancer: A Mechanism-Based Perspective

Round 1

Reviewer 1 Report

This is an extremely comprehensive review of TGFb, its downstream signaling, and an analysis of current TGFb inhibitors that have been used clinically or preclinically.  The author is clearly passionate about this area and shows an in-depth understanding which he conveys to the readers.  My only critique is the length of this article is more of a book chapter rather than a review; however, this should not limit its publication.  This is a very useful reference that investigators can call upon when looking into the use of TGFb inhibitors in cancer (and also for other diseases).  Below are three of several  points the author made that I found critical to moving this field forward and glad that he brought them to the readers attention:

·       Notably, most clinically available TGF-β antagonists block all three TGF-β isoforms, likely contributing to dose-limiting cardiotoxicities associated with  nonselective inhibition of multiple TGF-β isoforms, particularly TGF-βs 2 and 3 consistent with cardiac syndrome from mutations in TGF-βs 2 or 3 genes.

·       Many different types of TGF-β signaling inhibitors have been developed and co tinue to be in development, an increasing number of which target discreet components of 969 TGF-β signaling in cancers for a more targeted approach (Table 1).

·       11.1. Important Considerations:  When considering TGF-β inhibitor therapies for cancer patients, it is prudent to conduct a thorough evaluation of various factors to best tailor treatment strategies.

·       When gauging TGF-β isoforms, emphasis should be placed on measuring protein levels of TGF-β rather than solely relying on mRNA levels, as TGF-β isoforms undergo translational control

I only have a few minor formatting suggestions:

·       Page 10 – Figure 3 and its legend are separated spatially with main text.

·       Page 15 – Figure 4 legend needs to be aligned correctly below figure

·       Page 20 – Last paragraph is not aligned correctly with rest of text.

·       Page 27 – First row “none” should be capitalized  “None” to be consistent with other rows

·       Page 28 – Second row “none” should be capitalized “None” to be consistent with other rows

Author Response

Reviewer’s comments:  “This is an extremely comprehensive review of TGFb, its downstream signaling, and an analysis of current TGFb inhibitors that have been used clinically or preclinically.  The author is clearly passionate about this area and shows an in-depth understanding which he conveys to the readers.  My only critique is the length of this article is more of a book chapter rather than a review; however, this should not limit its publication.  This is a very useful reference that investigators can call upon when looking into the use of TGFb inhibitors in cancer (and also for other diseases).  Below are three of several points the author made that I found critical to moving this field forward and glad that he brought them to the readers attention:

• Notably, most clinically available TGF-β antagonists block all three TGF-β isoforms, likely contributing to dose-limiting cardiotoxicities associated with nonselective inhibition of multiple TGF-β isoforms, particularly TGF-βs 2 and 3 consistent with cardiac syndrome from mutations in TGF-βs 2 or 3 genes.
• Many different types of TGF-β signaling inhibitors have been developed and continue to be in development, an increasing number of which target discreet components of 969 TGF-β signaling in cancers for a more targeted approach (Table 1).
• 11.1. Important Considerations:  When considering TGF-β inhibitor therapies for cancer patients, it is prudent to conduct a thorough evaluation of various factors to best tailor treatment strategies.
• When gauging TGF-β isoforms, emphasis should be placed on measuring protein levels of TGF-β rather than solely relying on mRNA levels, as TGF-β isoforms undergo translational control”

Author’s response:  Thanks for sharing the many strengths of this manuscript.

 Reviewer’s comment: “I only have a few minor formatting suggestions:

• Page 10 – Figure 3 and its legend are separated spatially with main text.
• Page 15 – Figure 4 legend needs to be aligned correctly below figure
• Page 20 – Last paragraph is not aligned correctly with rest of text.
• Page 27 – First row “none” should be capitalized “None” to be consistent with other rows
• Page 28 – Second row “none” should be capitalized “None” to be consistent with other rows”

Author’s response: Thanks for pointing these to my attention. All the about formatting problems and more were corrected.

Reviewer 2 Report

Comments to Authors.

The review “Advances and Challenges in Targeting TGF-β Isoforms for  Therapeutic Intervention of Cancer: A Mechanism-Based Perspective” is a comprehensive literature review about TGF-β – targeted therapy and TGF-β isoforms functions.

The most interesting tasks for the pharmacologists in this review are refined approaches to enhance the effectiveness of TGF-β-targeted cancer therapies. The failure of different substances in clinical trials is also very important issue, in this review possible reasons are discussed.

To my opinion the review is too big and not well structured. For example, the chapter Smads and transcriptional control can be shortened and others molecular biological aspects can be shortened as the review have been submitted to Pharmaceuticals.

My proposals are listed below.

1.      In “Table 1. List of TGF-β Signaling Inhibitors Used in Preclinical and Clinical Studies” the data from preclinical and clinical studies are introduced, but there is no main conclusion in the table, just reference. It is not comfortable for the reader.  It is desirable to input the main result in the columns

2.       It is advisable to add a summary at the beginning so that the reader can conveniently see all the chapters at once.

3.       Fig. 8 is a print screen. Fig. 8 name is repeated

4.       TGF-β endogenous regulators are not mentioned. For example, steroid hormones.

5.       Chapters in bold  “Early times in TGF-β research”, “TGF-β Biosynthesis and Activation” etc. have no numbers. After chapter 2 numeration chapter 9 appears (page 14, line 582)

6.       Section 12. Summary and Future Projections is too big and vague, a stream of facts rather than conclusions. It is necessary to structure the summary and reduce the volume. Perhaps numbering of the main points is needed like highlights.

It is advisable to formulate the facts more briefly 

Author Response

Reviewer’s comments:

“The review “Advances and Challenges in Targeting TGF-β Isoforms for Therapeutic Intervention of Cancer: A Mechanism-Based Perspective” is a comprehensive literature review about TGF-β – targeted therapy and TGF-β isoforms functions.

 The most interesting tasks for the pharmacologists in this review are refined approaches to enhance the effectiveness of TGF-β-targeted cancer therapies. The failure of different substances in clinical trials is also very important issue, in this review possible reasons are discussed.

To my opinion the review is too big and not well structured. For example, the chapter Smads and transcriptional control can be shortened, and others molecular biological aspects can be shortened as the review have been submitted to Pharmaceuticals.”

Author’s response: I shortened the section on Smads a bit and included recent references in Pharmaceuticals to help the readers. I compared the molecular biology aspects in my manuscript with those in manuscripts submitted to Pharmaceuticals and decided to keep most of that text in my manuscript to help readers. The other manuscripts in this special issue either do not contain enough information on the molecular biology aspects or include too great a complexity, and are likely to confuse a reader new to the field.

Reviewer’s comment: “My proposals are listed below.”

1. Reviewer’s comment: “In “Table 1. List of TGF-β Signaling Inhibitors Used in Preclinical and Clinical Studies” the data from preclinical and clinical studies are introduced, but there is no main conclusion in the table, just reference. It is not comfortable for the reader.  It is desirable to input the main result in the columns.”

Author’s response: In compliance with this comment, I extensively revised that table and included the main conclusions of those studies. To do so, I reformatted that table and reduced the font size to make sufficient space to include that information. I also generated a list of abbreviations with that table.

2. Reviewer’s comment:”It is advisable to add a summary at the beginning so that the reader can conveniently see all the chapters at once.”

Author’s response: In compliance, I significantly changed the introduction and added a summary in the introduction section.

3. Reviewer’s comment: “Fig. 8 is a print screen. Fig. 8 name is repeated.”

Author’s response: This duplication was deleted.

4. Reviewer’s comment: “TGF-β endogenous regulators are not mentioned. For example, steroid hormones.”

Author’s response: I have now included endogenous regulators of TGF-βs expression in the section on TGF-β ligands.

5. Reviewer’s comment: “Chapters in bold “Early times in TGF-β research”, “TGF-β Biosynthesis and Activation” etc. have no numbers. After chapter 2 numeration chapter 9 appears (page 14, line 582).”

Author’s response: I submitted this manuscript with numbers in all those sections.  However, somehow those numbers were erroneously deleted during the process of reformatting this manuscript by the journal’s editorial office. I added all those numbers back in the revised manuscript.

6. Reviewer’s comment: “Section 12. Summary and Future Projections is too big and vague, a stream of facts rather than conclusions. It is necessary to structure the summary and reduce the volume. Perhaps numbering of the main points is needed like highlights.”

Author’s response: In compliance, the Summary and Future Projections section was extensively revised and focused accordingly. It is now only one page long.

 Comments on the Quality of English Language

Reviewer’s comment: “It is advisable to formulate the facts more briefly.”

Author’s response: The manuscript was extensively modified to improve clarity and brevity.

Reviewer 3 Report

This is a well done didactic paper explaining the prominent roles of TGFbeta(s) in cancer. It will be of value to all researchers in cancer growth and others trying to understand our complex cytokine signaling system. The subject might be too large for a single paper ? The author could consider breaking this major work up into a series of five papers, TGF-beta1, beta2, beta3, the various TGF inhibitors and a short overview paper summarizing the differences ? Each of those would be a major effort for readers. 

Assigning this paper in a graduate study class would occupy much of the semester to read and understand in detail.

Slight proofreading is required to correct minor typos [ex. Line 1005].

Figure 1 requires a legend with all abbreviations used in the schematic [drawing] spelled out. Also maybe better to show inside the drawing a note showing the barred red line with = inhibits, and show a black arrow with a note = stimulates. The figure otherwise is superb. 

Fix Line 64, double space.

Line 67 Probably ok to use “HPLC column,” without defining, but line 72 “125I-EGF to specific,” does require defining as EGFR has several ligands - epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, amphiregulin, epiregulin, etc.

Re line 87, it isn’t clear to me that rat 3T3 fibroblasts are malignant or not. Can you make this clear in the manuscript ?

Line 134, right, we do. “Today, a significant portion of investigators engaged in TGF-β research tend to erroneously assume a uniform functionality among all its three isoforms.” Can you include this comment in Abstract ?

Line 139. Do the authors think it worth specifying “a similar search on TGF-β1 and TGF-β without further specification identified. . .” ?

Line 140. Am I correct then in thinking both 1] the differences between the TGF-beta isoforms and, 2] the principle differences in resultant signaling effect depending on milieu and what co-signaling might be active, together makes much previous TGF-beta research not useful ? If yes, did you want to explicitly state that ?

Line 147, humans and rodents are not that distant. We are a lot closer to rodents than to dogs, bears, felines, f. Ex.

Line 163 would it help to say “each of the three TGF-β isoforms…” to remind readers ?

Line 164 is remarkable. Would the authors like to include their sentence in the Abstract ? [“Each of the three TGF-β isoforms is encoded on a different gene, is located on a different chromosome, and has a unique set of gene promoter and enhancer elements”]

Fig 2, to what is SMAD 4 pointing ? [lower left corner].

Re. fig 4, it might help readers to make arrows and barred lines [inhibitory] thicker, clearer. Your paper is heavy, so anything you can do to ease the reader’s burden is welcome.

Would the authors think it useful to discuss pirfenidone ?  This might be of interest in the fibrosis section.

It would help readers if the authors could break up their larger paragraphs. This also is warranted in that most of the long paragraphs contain different enough subjects to warrant breaking them up.

A dense and complicated paper like this requires a Table or box or preface listing the abbreviations. Ideally that Table would function as a glossary, giving a short description of the item. I understand that doing this will lengthen an already long paper. But this paper could be considered a major treatise on their subject.

Line 764. The author reverts here to TGF-beta, not specifying 1, 2 or 3. Perhaps the misunderstanding is mine but hasent the author established that there are deep and fundamental differences between the three TGF-betas ? It seems prior to that paragraph that the concept of “TGF-beta” becomes useless given the disparity of effects between 1, 2, and 3. Am I correct in this understanding ?

Throughout the manuscript, if my understanding is correct, the term TGF-beta should always be  given as 1, 2, or 3 or left out. Given the disparity of effects, disparity of promoters, disparity of signaling, does it tell us anything useful to just say TGF-beta ? 

This is a well done didactic paper explaining the prominent roles of TGFbeta(s) in cancer. It will be of value to all researchers in cancer growth and others trying to understand our complex cytokine signaling system. The subject might be too large for a single paper ? The author could consider breaking this major work up into a series of five papers, TGF-beta1, beta2, beta3, the various TGF inhibitors and a short overview paper summarizing the differences ? Each of those would be a major effort for readers. 

Assigning this paper in a graduate study class would occupy much of the semester to read and understand in detail.

Slight proofreading is required to correct minor typos [ex. Line 1005].

Figure 1 requires a legend with all abbreviations used in the schematic [drawing] spelled out. Also maybe better to show inside the drawing a note showing the barred red line with = inhibits, and show a black arrow with a note = stimulates. The figure otherwise is superb. 

Fix Line 64, double space.

Line 67 Probably ok to use “HPLC column,” without defining, but line 72 “125I-EGF to specific,” does require defining as EGFR has several ligands - epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, amphiregulin, epiregulin, etc.

Re line 87, it isn’t clear to me that rat 3T3 fibroblasts are malignant or not. Can you make this clear in the manuscript ?

Line 134, right, we do. “Today, a significant portion of investigators engaged in TGF-β research tend to erroneously assume a uniform functionality among all its three isoforms.” Can you include this comment in Abstract ?

Line 139. Do the authors think it worth specifying “a similar search on TGF-β1 and TGF-β without further specification identified. . .” ?

Line 140. Am I correct then in thinking both 1] the differences between the TGF-beta isoforms and, 2] the principle differences in resultant signaling effect depending on milieu and what co-signaling might be active, together makes much previous TGF-beta research not useful ? If yes, did you want to explicitly state that ?

Line 147, humans and rodents are not that distant. We are a lot closer to rodents than to dogs, bears, felines, f. Ex.

Line 163 would it help to say “each of the three TGF-β isoforms…” to remind readers ?

Line 164 is remarkable. Would the authors like to include their sentence in the Abstract ? [“Each of the three TGF-β isoforms is encoded on a different gene, is located on a different chromosome, and has a unique set of gene promoter and enhancer elements”]

Fig 2, to what is SMAD 4 pointing ? [lower left corner].

Re. fig 4, it might help readers to make arrows and barred lines [inhibitory] thicker, clearer. Your paper is heavy, so anything you can do to ease the reader’s burden is welcome.

Would the authors think it useful to discuss pirfenidone ?  This might be of interest in the fibrosis section.

It would help readers if the authors could break up their larger paragraphs. This also is warranted in that most of the long paragraphs contain different enough subjects to warrant breaking them up.

A dense and complicated paper like this requires a Table or box or preface listing the abbreviations. Ideally that Table would function as a glossary, giving a short description of the item. I understand that doing this will lengthen an already long paper. But this paper could be considered a major treatise on their subject.

Line 764. The author reverts here to TGF-beta, not specifying 1, 2 or 3. Perhaps the misunderstanding is mine but hasent the author established that there are deep and fundamental differences between the three TGF-betas ? It seems prior to that paragraph that the concept of “TGF-beta” becomes useless given the disparity of effects between 1, 2, and 3. Am I correct in this understanding ?

Throughout the manuscript, if my understanding is correct, the term TGF-beta should always be  given as 1, 2, or 3 or left out. Given the disparity of effects, disparity of promoters, disparity of signaling, does it tell us anything useful to just say TGF-beta ? 

Author Response

Comments by Reviewer 3:

“This is a well-done didactic paper explaining the prominent roles of TGFbeta(s) in cancer. It will be of value to all researchers in cancer growth and others trying to understand our complex cytokine signaling system. The subject might be too large for a single paper ? The author could consider breaking this major work up into a series of five papers, TGF-beta1, beta2, beta3, the various TGF inhibitors and a short overview paper summarizing the differences ? Each of those would be a major effort for readers. 

 Assigning this paper in a graduate study class would occupy much of the semester to read and understand in detail.

 Slight proofreading is required to correct minor typos [ex. Line 1005].”

Author’s response: This manuscript was thoroughly proofread for typos.

Reviewer’s comment: “Figure 1 requires a legend with all abbreviations used in the schematic [drawing] spelled out. Also maybe better to show inside the drawing a note showing the barred red line with = inhibits, and show a black arrow with a note = stimulates. The figure otherwise is superb. “

Author’s response: The legend of Figure 1 now includes abbreviations used in the drawing and within the figure itself a note to clarify stimulatory and inhibitory effects.

Reviewer’s comment: “ Fix Line 64, double space.”

Author’s response: This double space and all other double spaces in the text are now fixed. All are highlighted.

Reviewer’s comment: “Line 67 Probably ok to use “HPLC column,” without defining, but line 72 “125I-EGF to specific,” does require defining as EGFR has several ligands - epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, amphiregulin, epiregulin, etc.”

         Author’s response: Those ligands have now been included.

Reviewer’s comment: “Re line 87, it isn’t clear to me that rat 3T3 fibroblasts are malignant or not. Can you make this clear in the manuscript?”

       Author’s response: This has been now clarified in the text. Rat 3T3 fibroblasts are non-malignant.

Reviewer’s comment: “Line 134, right, we do. “Today, a significant portion of investigators engaged in TGF-β research tend to erroneously assume a uniform functionality among all its three isoforms.” Can you include this comment in Abstract?”

      Author’s response:  A modified version of this was incorporated in the abstract.

Reviewer’s comment: “Line 139. Do the authors think it worth specifying “a similar search on TGF-β1 and TGF-β without further specification identified. . .” ?”

             Author’s response: I believe it is important to share it for proper comparison.

Reviewer’s comment: “Line 140. Am I correct then in thinking both 1] the differences between the TGF-beta isoforms and, 2] the principle differences in resultant signaling effect depending on milieu and what co-signaling might be active, together makes much previous TGF-beta research not useful ? If yes, did you want to explicitly state that ?”

Author’s response:  In numerous studies involving the treatment of cells solely with TGF-β1, researchers often refer to it simply as TGF-β, potentially leading to confusion. It's worth noting that the downstream effects of signaling induced by TGF-β1, TGF-β2, and TGF-β3 tend to be comparable, if not indistinguishable. Consequently, there is a prevalent tendency among investigators to streamline their language by referring to "TGF-β signaling" for the sake of simplicity. However, this could be misleading, particularly in certain conditions in which there are differences in the function of the isoforms.  I have therefore edited this manuscript more thoroughly to indicate the specific TGF-β isoform used in the given study. Interestingly, some studies do not even specify the TGF-β isoform used in their methods section. One can only presume that they used TGF-β1 and instead wrote TGF-β for simplicity. 

Reviewer’s comment: “Line 147, humans and rodents are not that distant. We are a lot closer to rodents than to dogs, bears, felines, f. Ex.”

            Author’s response: Thanks for raising this issue. I changed that comparison between humans and chickens (separated by >300,000 years of divergent evolution).  Taken from the revised text:  “Notably, an exceptional feature of each TGF-β isoform is its remarkably evolutionarily preserved amino acid (aa) sequence, with a near-perfect amino acid sequence identity between distant relatives such as humans and chickens; two-way BLAST alignment of the mature sequence (last 112 aa) of TGF-β2 in Homo sapiens (Accession# NM_001135599.4) with that of Gallus gallus (Accession # NM_001031045.4) is 99.1% identical, with only one conserved substitution.”

Reviewer’s comment: “Line 163 would it help to say “each of the three TGF-β isoforms…” to remind readers ? “

Author’s response: Yes, it would help for the context of the section on TGF-β ligands.

Reviewer’s comment: “Line 164 is remarkable. Would the authors like to include their sentence in the Abstract? [“Each of the three TGF-β isoforms is encoded on a different gene, is located on a different chromosome, and has a unique set of gene promoter and enhancer elements”]”

 Author’s response: The abstract already exceeds the recommended length stipulated by the journal's guidelines. Thus, I included a shorter sentence for a similar message: Each TGF-β isoform is encoded on a separate gene with unique regulatory elements.

Reviewer’s comment: “Fig 2, to what is SMAD 4 pointing ? [lower left corner].”

 Author’s response: Thanks for sharing this ambiguity. I have clarified this with an arrow.

Reviewer’s comment: “Re. fig 4, it might help readers to make arrows and barred lines [inhibitory] thicker, clearer. Your paper is heavy, so anything you can do to ease the reader’s burden is welcome.”

 Author’s response:  The arrows and barred lines were thickened to improve readability.

Reviewer’s comment: “Would the authors think it useful to discuss pirfenidone ?  This might be of interest in the fibrosis section.”

 Author’s response: Thanks for suggesting discussing this. Due to the enormous length of this manuscript, I decided to not include this. Instead, several references were added to the section on fibrosis, one of which includes pirfenidone.

Reviewer’s comment: “It would help readers if the authors could break up their larger paragraphs. This also is warranted in that most of the long paragraphs contain different enough subjects to warrant breaking them up.”

 Author’s response: In compliance with this reviewer’s suggestion, I reviewed all the seemingly large paragraphs and broke them into two or more where appropriate or helpful.

Reviewer’s comment: A dense and complicated paper like this requires a Table or box or preface listing the abbreviations. Ideally that Table would function as a glossary, giving a short description of the item. I understand that doing this will lengthen an already long paper. But this paper could be considered a major treatise on their subject.

 Author’s response:  In compliance with this suggestion, I have included a preface of abbreviations.

Reviewer’s comment: “Line 764. The author reverts here to TGF-beta, not specifying 1, 2 or 3. Perhaps the misunderstanding is mine, but hasn’t the author established that there are deep and fundamental differences between the three TGF-betas? It seems prior to that paragraph that the concept of “TGF-beta” becomes useless given the disparity of effects between 1, 2, and 3. Am I correct in this understanding?

Throughout the manuscript, if my understanding is correct, the term TGF-beta should always be given as 1, 2, or 3 or left out. Given the disparity of effects, disparity of promoters, disparity of signaling, does it tell us anything useful to just say TGF-beta? “

 Author’s response:  In numerous studies involving the treatment of cells solely with TGF-β1, researchers often refer to it simply as TGF-β, potentially leading to confusion. It's worth noting that the downstream effects of signaling induced by TGF-β1, TGF-β2, and TGF-β3 tend to be comparable (although not identical), in several comparative studies. Consequently, there is a tendency of some investigators to streamline their language by referring to "TGF-β signaling" for the sake of simplicity. However, there is insufficient evidence that the signaling pathway of each isoform is the same, as differences in their biological responses have been reported.  Therefore, in compliance with your comment, I extensively modified the text of my manuscript to specify the particular isoform used or described when known. Most TGF-β studies both treated cells with TGF-β1 and studied response to only the TGF-β1 isoform, but reported their results as TGF-β instead of TGF-β1.  I surveyed the data in those studies and changed my writeup from TGF-β to TGF-β1 when appropriate.  I also included a section in the introduction to clarify these points.

Round 2

Reviewer 2 Report

Dear Authors,

The article have been sighnificantly improved and carefully revised.